Aphanamixoid A, a Potent Defensive Limonoid, with a New Carbon

ORGANIC LETTERS

Aphanamixoid A, a Potent Defensive Limonoid, with a New Carbon Skeleton from Aphanamixis polystachya

2012 Vol. 14, No. 10 2524–2527

Jie-Yun Cai,†,‡ Yu Zhang,† Shi-Hong Luo,† Duo-Zhi Chen,†,‡ Gui-Hua Tang,†,‡ Chun-Mao Yuan,† Ying-Tong Di,† Sheng-Hong Li,† Xiao-Jiang Hao,*,† and Hong-Ping He*,† State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, P. R. China, and Graduate School of Chinese Academy of Sciences, Beijing 100039, P. R. China [email protected]; [email protected] Received March 30, 2012

ABSTRACT

Aphanamixoid A (1), a limonoid with a new carbon skeleton, along with its biogenetically related limonoid aphanamixoid B (2), was isolated from the leaves and twigs of Aphanamixis polystachya. Their structures with the absolute stereochemistry were determined by spectroscopic analysis, X-ray crystallography and computational methods. The significant antifeedant activity of 1 against the generalist plant-feeding insect Helicoverpa armigera (EC50 = 0.015 μmol/cm2) suggested it may be a potent defensive component of A. polystachya.

Limonoids, a series of structurally diverse and highly oxygenated tetranortriterpenoids mainly found in the family of Meliaceae, have been attracting continuous attention from biogenetic and synthetic points of view.1 In recent years, a number of limonoids have still been †

Kunming Institute of Botany. Graduate School of the Chinese Academy of Sciences. (1) (a) Fang, X.; Di, Y. T.; Hao, X. J. Curr. Org. Chem. 2011, 15, 1363–1391. (b) Tan, Q. G.; Luo, X. D. Chem. Rev. 2011, 111, 7437–7522. (c) Fang, X.; Di, Y. T.; He, H. P.; Liu, H. Y.; Zhang, Z.; Ren, Y. L.; Gao, Z. L.; Gao, S.; Hao, X. J. Org. Lett. 2008, 10, 1905–1908. (d) Han, M. L.; Zhang, H.; Yang, S. P.; Yue, J. M. Org. Lett. 2012, 14, 486–489. (e) Fukuzaki, T.; Kobayashi, S.; Hibi, T.; Ikuma, Y.; Ishihara, J.; Kanoh, N.; Murai, A. Org. Lett. 2002, 4, 2877–2880. (f) Trudeau, S.; Morken, J. P. Org. Lett. 2005, 7, 5465–5468. (g) Zhang, Q.; Di, Y. T.; He, H. P.; Fang, X.; Chen, D. L.; Yan, X. H.; Zhu, F.; Yang, T. Q.; Liu, L. L.; Hao, X. J. J. Nat. Prod. 2011, 74, 152–157. (h) Yin, J. L.; Di, Y. T.; Fang, X.; Liu, E. D.; Liu, H. Y.; He, H. P.; Li, S. L.; Li, S. F.; Hao, X. J. Tetrahedron Lett. 2011, 52, 3083–3085. (2) (a) Wang, X. N.; Yin, S.; Fan, C. Q.; Wang, F. D.; Lin, L. P.; Ding, J.; Yue, J. M. Org. Lett. 2006, 8, 3845–3848. (b) Murphy, B. T.; Brodie, P.; Slebodnick, C.; Miller, J. S.; Birkinshaw, C.; Randrianjanaka, L. M.; Andriantsiferana, R.; Rasamison, V. E.; TenDyke, K.; Suh, E. M.; Kingston, D. G. I. J. Nat. Prod. 2008, 71, 325–329. (c) Ning, J.; Di, Y. T.; Fang, X.; He, H. P.; Wang, Y. Y.; Li, Y.; Li, S. L.; Hao, X. J. J. Nat. Prod. 2010, 73, 1327–1331. ‡

10.1021/ol3008149 r 2012 American Chemical Society Published on Web 04/27/2012

isolated by several research groups, a few of which exhibited biological activities including cytotoxic,2 antimalarial,3 insect antifeedant,4 insecticidal,4a,5 and insect growth regulatory4b activities. The plant Aphanamixis polystachya (Wall.) R. N. Parker (Meliaceae), a timber tree, is mainly distributed in the tropical areas of Asia, such as India, Malaysia, Indonesia, and southern China.6 Previous chemical studies on this (3) (a) Yin, S.; Wang, X. N.; Fan, C. Q.; Liao, S. G.; Yue, J. M. Org. Lett. 2007, 9, 2353–2356. (b) Hay, A. E.; Ioset, J. R.; Ahua, K. M.; Diallo, D.; Brun, R.; Hostettmann, K. J. Nat. Prod. 2007, 70, 9–13. (4) (a) Isman, M. B.; Koul, O.; Luczynski, A.; Kaminski, J. J. Agric. Food Chem. 1990, 38, 1406–1411. (b) Fowles, R.; Mootoo, B.; Ramsewak, R.; Khan, A.; Ramsubhag, A.; Reynolds, W.; Nair, M. Pest Manage. Sci. 2010, 66, 1298–1303. (c) Nakatani, M.; Abdelgaleil, S. A. M.; Kurawaki, J.; Okamura, H.; Iwagawa, T.; Doe, M. J. Nat. Prod. 2001, 64, 1261–1265. (d) Koul, O.; Daniewski, W. M.; Multani, J. S.; Gumulka, M.; Singh, G. J. Agric. Food Chem. 2003, 51, 7271–7275. (5) (a) Xu, H.; Zhang, J. L. Bioorg. Med. Chem. Lett. 2011, 21, 1974– 1977. (b) Ge, Y. H.; Zhang, J. X.; Mu, S. Z.; Chen, Y.; Yang, F. M.; Lu, Y.; Hao, X. J. Tetrahedron 2012, 68, 566–572. (6) Chen, S. K.; Chen, B. Y.; Li, H. Flora Reipublicae Popularis Sinicae (Zhongguo Zhiwu Zhi); Science Press: Beijing, China, 1997; Vol. 43, pp 239
240.

plant have resulted in the isolation of a few new limonoids; however, no significant bioactivity has been found from those compounds.7 In the current study, a new limonoid with potent antifeedent activity, aphanamixoid A (1), was isolated from the leaves and twigs of A. polystachya collected in the Yunnan province of China. 1 could be derived from a new biogenetically related limonoid, aphanamixoid B (2), via the unique cleavage of a C-9
C-10 bond as well as the formation of a C-2
C-30 bond by means of 3,3-rearrangement. In this paper, we report the isolation, structure elucidation, plausible biogenetic pathway, and the bioactivities of aphanamixoids A (1) and B (2).

Table 1. 1H and 13C NMR Data for 1 and 2 in CDCl3 1 δCa 1 2 3 4 5 6a 6b 7 8 9 10 11R 11β 12 13 14 15 16R

120.2 (d) 43.7 (d) 172.7 (s) 81.4 (s) 47.8 (d) 34.4 (t)

δH (mult; J, Hz)a

2 δ Ca

5.18 (s) 3.42 (br s)

81.6 (d) 38.2 (t) 170.3 (s) 84.8 (s) 2.74 (dd, 6.2, 4.0) 48.6 (d) 2.23 (dd, 17.0, 4.0) 33.4 (t) 2.82 (dd, 17.0, 6.2) 173.4 (s) 173.7 (s) 131.0 (s) 140.3 (s) 125.0 (d) 5.50 (d, 3.4) 55.6 (d) 137.8 (s) 50.5 (s) 29.9 (t) 2.22 (m) 79.3 (d) 2.47 (m) 77.6 (d) 5.07 (dd, 10.0, 6.1) 75.8 (d) 50.2 (s) 51.7 (s) 147.6 (s) 149.4 (s) 121.7 (d) 5.65 (s) 122.9 (d) 37.7 (t) 2.58 (m, 2H) 37.8 (t)

16β 8

Aphanamixoid A (1) was obtained as colorless crystals (in acetone). Its molecular formula, C29H36O7, was established from the quasi-molecular ion peak at m/z 519.2361 [M þ Na]þ (calcd 519.2358, C29H36O7Na) in the positive HRESIMS, which indicated 12 degrees of unsaturation. UV absorption at 242 nm (3.44) indicated the presence of conjugated double bonds. IR peaks at 1732 and 1717 cm
1 as well as 13C NMR signals at δ 173.4, 172.7, and 170.9 revealed three ester carbonyl groups. Besides a methoxy group (δH 3.70; δC 52.1) and an acetyl group (δH 1.90; δC 21.3, 170.9), 1 contained 26 carbons, including a β-furan ring (δH 6.32, 7.23, 7.35; δC 111.6, 140.0, 142.1) and four tertiary methyl groups (δH 0.90, 1.36, 1.61, 1.78). The above evidence suggested that 1 was a tetranortriterpenoid. Furthermore, apart from the five double bonds and three carbonyl groups, the remaining four degrees of unsaturation indicated 1 to be tetracyclic system. Extensive comparison of 1H and 13C NMR data with those of a known limonoid, munronoid B, suggested both (7) (a) Yang, S. P.; Chen, H. D.; Liao, S. G.; Xie, B. J.; Miao, Z. H.; Yue, J. M. Org. Lett. 2011, 13, 150–153. (b) Zhang, Y.; Wang, J. S.; Wang, X. B.; Wei, D. D.; Luo, J. G.; Luo, J.; Yang, M. H.; Kong, L. Y. Tetrahedron Lett. 2011, 52, 2590–2593. (c) Zhang, H.; Chen, F.; Wang, X.; Wu, D.; Chen, Q. Magn. Reson. Chem. 2007, 45, 189–192. (d) Sadhu, S. K.; Phattanawasin, P.; Choudhuri, M. S. K.; Ohtsuki, T.; Ishibashi, M. J. Nat. Med. 2006, 60, 258–260. (e) Mulholland, D. A.; Naidoo, N. Phytochemistry 1999, 51, 927–930. (8) Aphanamixoid A (1): colorless crystals (in acetone); mp 148
150 °C; HRESIMS at m/z 519.2361 [M þ Na]þ (calcd 519.2358, C29H36O7Na); [R]D14.0 = þ39.9° (c 0.260, MeOH); UV (MeOH) λmax (log ε) 242 (3.44), 202 (3.39); CD (0.000511 M, MeOH) λmax (Δε) 231 (1.14); IR νmax (KBr) cm
1 1732, 1717, 1265, 1255, and 1245; 1H and 13C NMR data, see Table 1. (9) Flack, H. D. Acta Crystallogr., Sect. A 1983, 39, 876–881. (10) Aphanamixoid B (2): colorless amorphous powder; mp 170
171 °C; HRESIMS at m/z 535.2314 [M þ Na]þ (calcd 535.2307, C29H36O8Na); [R]D14.3 = þ81.8° (c 0.140, MeOH); UV (MeOH) λmax (log ε) 210 (3.06), 202 (3.07); CD (0.000342 M, MeOH) λmax (Δε) 236 (1.81), 205 (0.56); IR (KBr) νmax 2957, 2930, 1739, 1373, 1235, and 1030 cm
1; 1H and 13C NMR data, see Table 1. Org. Lett., Vol. 14, No. 10, 2012

17 18 19 20 21 22 23 28 29 30a 30b 12-OAc 7-OMe

46.4 (d) 13.2 (q) 25.5 (q) 124.6 (s) 140.0 (d) 111.6 (d) 142.1 (d) 28.8 (q) 25.7 (q) 34.7 (t) 170.9 (s) 21.3 (q) 52.1 (q)

3.15 (dd, 9.9, 8.7) 0.90 (s) 1.78 (s)

47.2 (d) 15.1 (q) 20.4 (q) 124.3 (s) 7.23 (s) 139.9 (d) 6.32 (s) 111.1 (d) 7.35 (br s) 142.4 (d) 1.36 (s) 30.6 (q) 1.61 (s) 27.7 (q) 2.30 (dd, 14.2, 9.1) 118.8 (t) 2.97 (dd, 14.2, 3.7) 170.8 (s) 1.90 (s) 21.2 (q) 3.70 (s) 52.3 (q)

δH (mult; J, Hz)b 4.07 (dd, 10.9, 4.8) 2.87 (m, 2H)

2.90 (dd, 6.9, 3.8) 2.41 (dd, 17.0, 6.9) 3.07 (dd, 17.0, 3.8)

3.06 (d, 8.2) 4.11 (dd, 9.7, 8.2) 5.35 (d, 9.7)

5.76 (t, 3.0) 2.39 (ddd, 16.5, 10.5, 3.0) 2.62 (ddd, 16.5, 8.4, 3.0) 3.30 (dd, 10.5, 8.4) 0.80 (s,CH3) 1.17 (s, CH3) 7.19 (s) 6.24 (s) 7.33 (br s) 1.40 (s, CH3) 1.45 (s, CH3) 5.12 (s) 5.40 (s) 1.95 (s, CH3) 3.76 (s, CH3)

a1 H NMR spectra were recorded at 400 MHz and 13C NMR spectrum at 100 MHz. b 1H NMR spectrum was recorded at 500 MHz.

compounds shared the same A, D, and E ring systems, as further confirmed by 2D NMR studies. Furthermore, the replacement of a C-30 sp2 methylene signal in munronoid B5b with a sp3 methylene signal (δH 2.30, 2.97; δC 34.7) in 1, as well as the striking presence of two double bonds (Δ8(9) and Δ1(10)) in 1 implied that the methylene group (C-30) might be the core linkage between rings A and C instead of the usual connectivity via C-9 and C-10. (11) (a) Lidert, Z.; Taylor, D. A. H.; Thirugnanam, M. J. Nat. Prod. 1985, 48, 843–845. (b) Sarker, S. D.; Savchenko, T.; Whiting, P.; Sik, V.; Dinan, L. Arch. Insect Biochem. Physiol. 1997, 35, 211–217. (12) (a) Zhang, Y.; Di, Y. T.; Zhang, Q.; Mu, S. Z.; Tan, C. J.; Fang, X.; He, H. P.; Li, S. L.; Hao, X. J. Org. Lett. 2009, 11, 5414–5417. (b) Zhang, Q.; Di, Y. T.; Li, C. S.; Fang, X.; Tan, C. J.; Zhang, Z.; Zhang, Y.; He, H. P.; Li, S. L.; Hao, X. J. Org. Lett. 2009, 11, 2357–2359. (13) Frisch, M. J. et al. Gaussian 03, Revision D.01; Gaussian, Inc.: Wallingford, CT, 2005 (see the Supporting Information for full reference). (14) (a) Tan, C. J.; Di, Y. T.; Wang, Y. H.; Zhang, Y.; Si, Y. K.; Zhang, Q.; Gao, S.; Hu, X. J.; Fang, X.; Li, S. F.; Hao, X. J. Org. Lett. 2010, 12, 2370–2373. (b) Wang, L.; He, H. P.; Di, Y. T.; Zhang, Y.; Hao, X. J. Tetrahedron Lett. 2012, 53, 1576–1578. 2525

Figure 2. Single crystal X-ray structure of 1.

Figure 1. Key 1H
1H COSY (a: ;), HMBC (a: f (red)), and ROESY (b: T (red)) correlations of 1.

HMBC correlations of H3-19/C-1 (δC 120.2), C-5 (δC 47.8), and C-10 (δC 137.8), H-5 (δH 2.74)/C-5 and C-10 as well as the downfield-shifted hydrogen resonances of H-1 (δH 5.18) and 1H
1H COSY correlations (H-5
H-6) indicated the location of a double bond between C-1 and the quaternary carbon atom C-10, which also suggested the cleavage of C-9 and C-10. The 1H
1H COSY correlations (H-1
H-2
H2-30) and the HMBC correlations of H2-30/ C-1, C-2, C-3, C-8 (δC 131.0), and C-9 (δC 125.0) confirmed that C-2 and C-8 were linked through C-30 as shown in Figure 1. HMBC correlations of H-30/C-14 (δC 147.6), H-15 (δH 5.65)/C-8, and C-14 indicated the presence of Δ14(15) double bond, which was conjugated with Δ9(8) double bond. Additionally, the acetoxy group was located at C-12 by the HMBC cross signal H-12/C-12-OAc, and the HMBC correlations of H3-28/C-3 together with the downfield-shifted carbon resonances of C-3 and C-4 definitely indicated the linkage of C-3 and C-4 via an oxygen atom to form the unsaturated lactone ring A. Thus, the aforementioned data suggested a unique ring A, B-seco limonoid with a unique C-2
C-30 bond, and the planar structure of 1 was established as shown in Figure 1. The relative stereochemistry of 1 was determined by ROESY spectrum (Figure 1b). Furthermore, the successful performance of the X-ray diffraction experiment with Cu KR radiation confirmed the proposed structure and also

(15) (a) Luo, S. H.; Luo, Q.; Niu, X. M.; Xie, M. J.; Zhao, X.; Schneider, B.; Gershenzon, J.; Li, S. H. Angew. Chem., Int. Ed. 2010, 49, 4471–4475. (b) Luo, S. H.; Weng, L. H.; Xie, M. J.; Li, X. N.; Hua, J.; Zhao, X.; Li, S. H. Org. Lett. 2011, 13, 1864–1867. 2526

allowed unambiguous assignment of the absolute configuration of 1 as drawn [Flack parameter: 0.1(2)]9 (Figure 2). Aphanamixoid B (2)10 was obtained as colorless amorphous powder. The molecular formula was determined as C29H36O8 with 12 degrees of unsaturation deduced by HRESIMS at m/z 535.2314 [M þ Na] þ (calcd 535.2307, C29H36O8Na). The IR absorption band at 1739 cm
1 indicated the presence of ester carbonyl groups. The observation for a β-furan ring (δH 6.24, 7.19, 7.33; δC 111.1, 139.9, 142.4), a methoxy group (δH 3.76; δC 52.3), four tertiary methyl groups (δH 0.80, 1.17, 1.40, 1.45), and a characteristic exocyclic double bond (δH 5.12, 5.40) in the 1 H and 13C NMR spectra of 2 strongly suggested that 2 was a prieurianin-type limonoid.4d,11 The 1H and 13C NMR spectral data of 2 showed close similarity to those of the reported compound, munronoid A,5b except for the absence of an acetyl group, as well as the presence of an additional oxygenated methine (δH 4.11). Compared with munronoid A, the observed significant downfield shifts of C-1 (δC 81.6) and C-11 (δC 79.3) together with the strong HMBC correlation (Figure 3a) connected via an oxygen atom and formed a tetrahydrofuran ring. The structure of 2 was confirmed by 2D NMR (HSQC, HMBC, 1H
1H COSY, and ROESY) experiments (Figure 3). The absolute configuration of aphanamixoid B (2) was assigned using the quantum chemical method. The optical rotation (OR) value of 2 was calculated using density functional theory (DFT) methods12 in the Gaussian 03 program package.13 The “self-consistent reaction field” method (SCRF) was employed to perform the OR calculation of the most stable conformer of 2 in MeOH solution at the B3LYP/6-31G (d,p) level. The calculated OR value (þ91.4°) for 2 is close to its experimental value (þ81.8°), which suggested a reliable absolute configuration assignment for 2. In addition, its electronic circular dichroism (ECD)14 was also calculated on the Gaussian 03 program using TD-DFT-B3LYP/6-31G(d,p) level,13 which showed a good agreement with those of experimentally recorded Org. Lett., Vol. 14, No. 10, 2012

linkage followed by reduction of 2 formed i, and then dehydration to yield the key intermediate ii, which produced 1 by means of 3,3-rearrangement, as shown in Scheme 1.

Scheme 1. Plausible Biosynthetic Pathway for 1

Figure 3. Key 1H
1H COSY (a: ;), HMBC (a: f (red)), and ROESY (b: T (red)) correlations of 2.

The antifeedant activity of aphanamixoid A (1) against the larvae of two generalist insects, beet armyworm (Spodoptera exigua) and cotton bollworm (Helicoverpa armigera), were evaluated.15 The compound 1 exhibited a potent antifeedant activity with an EC50 value of 0.052 and 0.015 μmol/cm2, respectively. The results suggested a potent defensive role of 1 against herbivore enemies, while aphanamixoid B (2) showed moderate antifeedant activity against S. exigua at 2000 ppm with AFI of 17%. Acknowledgment. This research was supported financially by the National Natural Science Foundation of China (30830114), the Ministry of Science and Technology (2009CB522300 and 2009CB940900), and the Yong Academic and Technical Leader Raising Foundation of Yunnan Province (2010CI047). We thank Dr. Xiao-Nian Li (Kunming Institute of Botany, CAS) for help with analysis of the X-ray data.

Figure 4. Comparison of the experimental CD and calculated CD spectrum of 2.

CD spectrum (Figure 4). Thus, the absolute configuration of 2 was unambiguously assigned as depicted. The biogenetic origin of aphanamixoid A (1) might be derived from aphanamixoid B (2). The cleavage of ether

Org. Lett., Vol. 14, No. 10, 2012

Supporting Information Available. 1D and 2D NMR spectra of 1 and 2, experimental procedures, plant material, bioactivity assay, the X-ray crystallographic data for 1, and computational calculations for 2. This material is available free of charge via the Internet at http://pubs.acs.org. The authors declare no competing financial interest.

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